Rectoanal incontinence (RI) is a major health concern because it is debilitating and affects a significant portion of the US population, especially the elderly. The long-term goal of our studies is to identify the molecular mechanisms underlying the pathophysiology for aging-associated internal anal sphincter dysfunction (AAID) and to identify therapeutic targets not only for RI but also for other AAID-associated debilitating rectoanal motility disorders such as recurrent anal fissures and hemorrhoids. The focus of the present application is to establish the role of two novel regulatory factors ? brain-derived neurotrophic factor (BDNF) and Toll-like receptors (TLRs) ? in the aging-associated remodeling of the IAS smooth muscle that leads to the pathophysiology of AAID and to identify novel therapeutic targets for this condition. BDNF and TLRs may exert their effects by regulating RhoA/ROCK (a major molecular determinant of IAS tone) directly, via GPCR, and via downstream transcriptional regulators. Our central hypothesis is that remodeling of the IAS smooth muscle during AAID is characterized by loss of differentiation characteristics, with dysregulated RhoA/ROCK-signaling primarily because of downregulated BDNF and upregulated TLRs. The objective of this grant is to establish that BDNF (originally known to be neuroprotective) and TLRs play important roles in the AAID by disrupting RhoA/ROCK signaling either directly, via GPCR (AT1-R and TXA2-R), or via downstream transcriptional regulators TRKB/HDAC and NF-?B/LMOD1/MYOCD, respectively. Following identification of these cellular pathways in AAID, we plan to determine whether AAID can be rescued by BNDF/HDAC activators, TLR/NF-?B inhibitors, and by LMOD1 and MYOCD upregulation. The proposed innovative studies utilize state-of-the art molecular approaches and are expected to identify the molecular pathways involved in AAID in relation to the novel regulators BDNF and TLRs in the remodeling of the IAS smooth muscle. In order to validate the animal model for the AAID in RI in the aging humans, we have added specific studies using human tissues from different age groups, and bioengineered IAS. Thus, these studies will have direct clinical relevance in advancing the understanding of the pathophysiology of AAID and identifying novel therapeutic targets for RI. RELEVANCE Rectoanal incontinence (RI) is a major health concern because it is debilitating and affects a significant portion of the US population, especially the elderly. The focus of the proposed studies is on the aging- associated internal anal sphincter (IAS) dysfunction, a primary component of RI. Although RI is multifactorial, aging-associated IAS dysfunction (AAID) is one of the leading contributors. Using model systems in animals and humans, we will identify intracellular signaling molecules for BDNF and TLRs in the pathophysiology and therapeutic targeting for RI in the elderly.